Activated protein c formulations

ABSTRACT

The invention relates to pharmaccutical compositions of activated protein C with a chelating agent. Preferably, the formulation contains activated protein C, a chelating agent, a bulking agent, a buffer, and/or a salt with a reconstituted pH between about 5.5 5 and about 6.5. Alternatively, the chelating agent is added to the diluent used with the activated protein C pharmaceutical composition. The aPC pharmaceutical compositions, formulations, and uses of the invention have improved in-use stability.

FIELD OF THE INVENTION

This invention is in the field of human medicine, particularly in thetreatment of vascular disorders with activated protein C. Morespecifically, the invention relates to pharmaceutical compositions,formulations, and uses of recombinant human activated protein C.

BACKGROUND OF THE INVENTION

Protein C is a serine protease and naturally occurring anticoagulantthat plays a role in the regulation of homeostasis by inactivatingFactors Va and VIIIa in the coagulation cascade. Protein C activationresults from removal of a dodecapeptide at the N-terminus of the heavychain, producing activated protein C (aPC) possessing enzymaticactivity.

In addition to the enzymatic activities of aPC within the bloodcoagulation cascade, aPC also can degrade. Activated protein Cdegradation can lead to variants with anticoagulant activity similar tothe full-length chain or to less active variants. According to Foster etal. (WO 91/09951), aPC can include active variants consisting ofdeletions from the full-length light chain. Foster indicated that suchvariants include from 149 to 152 amino acid residues and that theselight chain deletions do not substantially alter the activity.

In contrast to Foster et al., Applicants discovered that the 1-149 lightchain variant was less active while the 1-150, 1-151, and 1-152 variantswere active. This less active variant leads to decreased functionalityas an anticoagulant via diminished potency.

In order to administer aPC to patients, freeze dried formulations of aPCformulation are reconstituted with diluent, added to an intravenousinfusion solution, and administered via intravenous transfusion over thecourse of many hours. This aPC in-use solution has shown decreasedpotency levels due to the presence of the less active 1-149 aPCC-terminal light chain variant. Therefore, minimizing the level of the1-149 aPC C-terminal light chain variant degradation product isimportant in achieving a potent in-use aPC formulation.

Applicants discovered that the addition of a chelating agent to thediluent used with the aPC formulation or to the aPC formulation itselfimproves the solution stability of aPC. Linnau et al. (AU 9892449)teaches pharmaceutical preparations of at least two blood coagulationfactors, including protein C, and involves the use of a chelating agent.Premature thrombin formation in the preparation is prevented if calciumions are displaced by the presence of magnesium or by the addition of achelating agent, including EDTA, or related substances, such as citrate.Linnau et al. fail to teach preparations of activated protein C or theeffect that metal ions or chelating agents may have on the propensity ofactivated protein C to form truncated variants or on its stability.Thus, there remains a need in the art to prepare stable, potentformulations of activated protein C. Accordingly, these discoveriesallow the preparation of potent in-use aPC formulations to the healthcare provider that are suitable for administration to a patient in needthereof.

SUMMARY OF THE INVENTION

The invention provides a pharmaceutical composition comprising aPC and achelating agent. The pharmaceutical composition contains the chelatingagent.

The invention also provides a pharmaceutical composition comprising aPC,a diluent, and a chelating agent. The diluent is either a reconstitutiondiluent or an intravenous infusion solution wherein the diluent containsthe chelating agent.

The invention further provides a pharmaceutical composition in the formof a lyophilized formulation. These pharmaceutical compositions containaPC, a chelating agent, a bulking agent, a buffer, and/or a salt. Uponreconstitution, the pharmaceutical composition has a pH of about 5.5 to6.5.

The invention also provides a process for preparing a lyophilizedformulation of aPC, which comprises freeze drying a pharmaceuticalformulation containing activated protein C, a bulking agent, and achelating agent.

Also provided is a process of preparing a pharmaceutical solution ofaPC, which comprises reconstituting a lyophilized formulation containingactivated protein C with a diluent containing a chelating agent.

Further provided is a process of preparing a pharmaceutical solution ofaPC, which comprises reconstituting a lyophilized formulation containingactivated protein C and a bulking agent with a diluent containing achelating agent.

Also provided is a method of treating a patient in need thereof whichcomprises administering to the patient the pharmaceutical composition ofthe present invention.

Additionally provided is a use of the pharmaceutical composition of thepresent invention which comprises treating thrombotic disorders.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of the invention, as disclosed and claimed herein, thefollowing terms are as defined below.

aPC or activated protein C refers to activated protein C whetherrecombinant or plasma derived. aPC includes and is preferably humanactivated protein C although aPC may also include other species orderivatives of activated protein C. Examples of protein C derivativesare selected from the group consisting of Gerlitz, et al., U.S. Pat. No.5,453,373, and Foster, et al., U.S. Pat. No. 5,516,650.

APTT—activated partial thromboplastin time.

r-hPC—recombinant human protein C zymogen.

rhAPC—recombinant human activated protein C.

r-aPC—recombinant activated protein C produced by activating protein Czymogen in vitro or in vivo or by direct secretion of the activated formof protein C from procaryotic cells, eukaryotic cells, or transgenicanimals including, for example, secretion from human kidney 293 cells asa zymogen then purified and activated by techniques well known to theskilled artisan. Examples are demonstrated in Yan, U.S. Pat. No.4,981,952, and Cottingham, WO 97/20043.

Continuous infusion—continuing substantially uninterrupted theintroduction of a solution into a blood vessel for a specified period oftime.

Bolus injection—the injection of a drug in a defined quantity (called abolus) at once.

Suitable for administration—a lyophilized formulation or solution thatis appropriate to be given as a therapeutic agent.

Zymogen—protein C zymogen, as used herein, refers to secreted, inactiveforms, whether one chain or two chains, of protein C.

Pharmaceutically acceptable buffer—a pharmaceutically acceptable bufferis known in the art. Pharmaceutically acceptable buffers include sodiumphosphate, sodium citrate, sodium acetate, or TRIS.

Chelating agent—a pharmaceutically acceptable multidentate ligand whosemolecules can form several bonds to a single metal ion, creating a ring.

Diluent—a diluent optionally is a reconstitution diluent or anintravenous infusion solution. A reconstitution diluent is a solutionused to restore a lyophilized material to the liquid state. Anintravenous infusion solution is a solution used as a vehicle for theadministration of pharmaceutical compositions or formulations to apatient. A lyophilized formulation is reconstituted prior to itsaddition to the intravenous infusion solution. Some examples ofdiluents, as either reconstitution solutions or intravenous infusionsolutions, include 0.9% Sodium Chloride, Sodium Chloride with PotassiumChloride, Glucose and Sodium Chloride, 5% Dextrose, Lactated Ringers, 3%Sodium Chloride, Sterile Water for Injection, and Ringers Injection.

EDTA—Ethylenediaminetetraacetic acid that optionally is alone or part ofa salt complex. Preferred EDTA forms are selected from the groupconsisting of dipotassium edetate, disodium edetate, edetate calciumdisodium, sodium edetate, trisodium edetate, EDTA free acid, EDTAdisodium salt, EDTA disodium salt dihydrate, EDTA disodium-calcium salt,EDTA dipotassium salt, EDTA tripotassium salt EDTA sodium salt, EDTAtrisodium salt, EDTA tetrasodium salt hydrate, EDTA tetrasodium salttetrahydrate, EDTA ferric-sodium salt, EDTA ferric-sodium salt hydrate,and other salts and hydrates. The term edetate is an abbreviation forEthylenediaminetetraacetic acid.

Activated protein C is an antithrombotic agent with a wider therapeuticindex than available anticoagulants, such as heparin and the oralhydroxycoumarin type anticoagulants. Activated protein C is also usefulin treating thrombotic disorders. As an antithrombotic agent, aPC has aprofound effect on the treatment of a wide variety of acquired diseasestates involving intravascular coagulation, including thrombotic stroke,deep vein thrombosis, pulmonary embolism, peripheral arterialthrombosis, emboli originating from the heart or peripheral arteries,acute myocardial infarction, disseminated intravascular coagulation, andacute pre or postcapillary occlusions, including transplantations orretina thrombosis. Activated protein C is useful in treating an acquiredhypercoagulable state or acquired protein C deficiency associated withsepsis, transplantations, burns, pregnancy, major surgery, trauma, orARDS. The present formulation provides a method of treating patients byadministering a pharmaceutical composition described herein. The presentformulation further provides the use of a pharmaceutical compositiondescribed herein for treating patients afflicted by one or more of theseconditions.

The invention relates to stable formulations and uses of aPC. One stablelyophilized formulation would consist of aPC and a chelating agent.Additionally, a bulking agent is optionally added to the presentformulation and selected from the group consisting of mannitol,trehalose, raffinose, and sucrose for use in the lyophilizedformulation. Furthermore, the ionic strength is optionally controlledthrough the salt concentration of the solution. Pharmaceuticallyacceptable salts typically used to generate ionic strength include butare not limited to potassium chloride (KCl) and sodium chloride (NaCl).The lyophilized product is reconstituted with the appropriate diluent.Preferably, the resulting solution has a pH of about 5.5 to about 6.5.To maintain effective pH control, the aPC solution may contain apharmaceutically acceptable buffer. Representative buffer systemsinclude Tris-acetate, sodium citrate, and sodium phosphate. A preferableformulation use is a stable lyophilized aPC product that isreconstituted with a diluent containing a chelating agent. Also, apreferable formulation use is a stable lyophilized aPC product that isreconstituted with a diluent then added to an intravenous infusionsolution containing a chelating agent or to which a chelating agent willbe added.

The molecular interactions in a formulation between aPC, buffer, saltconcentration, pH, temperature, and bulking agents, are complex. Yet,the addition of a chelating agent to an aPC formulation with any of theaforementioned components or parameters or to the diluent used with theaPC formulation inhibits less active 1-149 aPC C-terminal light chainvariant degradation products. The formulations of the invention providestable, enzymatically active, aPC with reduced degradation. Theinvention has reduced or eliminated any increase in the amount of 1-149aPC C-terminal light chain variant. Preferably, the levels of 1-149 aPCC-terminal light chain variant do not increase by more than 5%. Morepreferably, 1-149 aPC C-terminal light chain variant levels do notincrease by more than 3%. Even more preferably, the levels of 1-149 aPCC-terminal light chain variant do not increase by more than 2%. Mostpreferably, the levels of 1-149 aPC C-terminal light chain variant donot increase by more than 1%. This stability is obtained through carefulcontrol of the processing and formulation conditions and by the additionof a chelating agent.

While the particular source of the aPC is not relevant to theoperability of the claimed invention, one illustrative source isdepicted in Preparations 1 and 2 below. Preferably the aPC is free ofother vitamin-K dependent proteins and factors such as protein S,protein Z, as well as factors II, VU, IX, and X. The formulationsdescribed herein are directed to a pharmaceutical product suitable foradministration to a patient, either directly, after freeze drying andreconstitution, or, optionally, after dilution in an intravenousinfusion solution.

The use of chelating agents for the invention provide a means forsequestering metals that would otherwise promote aPC degradationresulting in the less active 1-149 light chain variant. Representativechelating agents include dipotassium edetate, disodium edetate, edetatecalcium disodium, sodium edetate, trisodium edetate, EDTA free acid,EDTA disodium salt, EDTA disodium salt dihydrate, EDTA disodium-calciumsalt, EDTA dipotassium salt, EDTA tripotassium salt EDTA sodium salt,EDTA trisodium salt, EDTA tetrasodium salt hydrate, EDTA tetrasodiumsalt tetrahydrate, EDTA ferric-sodium salt, EDTA ferric-sodium salthydrate, and other salts and hydrates. The most preferred chelatingagent is EDTA disodium salt The skilled artisan will recognize that manyother chelating agents are available for use in the formulations of theinvention. The optimum concentration of chelating agent is dependent onthe amount of metals present in the diluent. Yet, as long as asufficient concentration of chelating agent is used to sequester allavailable metals, any excess of chelating agent beyond the necessaryamount will have no undue effect on the pharmaceutical composition orformulation. However, the skilled artisan will recognize that the upperrange for the amount of chelating agent used is within physiologicaltolerance. Primarily, the amount of chelating agent is determined basedon the aPC concentration. Preferably, the amount of chelating agent isfrom 1 μM to 10 mM. More preferably, the amount of chelating agent isfrom 20 μM to 5 mM. The preferred concentration of chelating agent whenusing 2.5 mg/mL aPC is 1 μM to 10 mM. A more preferred concentration ofchelating agent when using 2.5 mg/mL aPC is 20 μM to 5 mM. Even morepreferably, the concentration of chelating agent when using 2.5 mg/mLaPC is 50 μM to 1 mM. The most preferred concentration of chelatingagent when using 2.5 mg/mL aPC is 500 μM. Alternatively, the preferredconcentration of chelating agent when using 5 mg/mL aPC is 1 μM to 10mM. A more preferred concentration of chelating agent when using 5 mg/mLaPC is 20 μM to 5 mM. Even more preferably, the concentration ofchelating agent when using 5 mg/mL aPC is 500 μM to 3 mM. The mostpreferred concentration of chelating agent when using 5 mg/mL aPC is 1mM. Using a chelating agent with the diluent containing the formulationstabilizes the aPC for at least 24 hours at room temperature.

Preferred bulking agents in the formulation of aPC are sucrose,trehalose and raffinose. More preferred bulking agents are sucrose andraffinose. The most preferred bulking agent is sucrose. The amount ofbulking agent in the formulation is 1 part aPC to 1 to 10 parts bulkingagent on a weight-to-weight basis. Moreover, the bulking agentconcentration of the formulation is an important formulation variable ofthe freeze drying process. The optimum concentration of bulking agent isdependent on the amount of aPC and species of bulking agent selected.The preferred concentration of sucrose in the freezing solution is 10 to40 mg/mL. A more preferred concentration of sucrose is 15 to 30 mg/mL.The most preferred concentration of sucrose in the freezing solution is15 mg/mL in a formulation of aPC at 2.5 mg/mL. The most preferredconcentration of sucrose in the freezing solution is 30 mg/mL in aformulation of aPC at 5.0 mg/mL. The presence of the bulking agent inthe formulation of aPC offers increased chemical and physical stability.

Prior to freeze drying and upon reconstitution, it is preferable tomaintain the pH in the range of 5.5 to 6.5 to minimize solution stateautodegradation. The preferred pH of the formulation is a pH betweenabout pH 5.6 and about pH 6.4. More preferred is a pH between about 5.7to about 6.3. Even more preferred is a pH between about 5.8 to about6.2. Still even more preferred is a pH between about 5.9 to about 6.1.The most preferred pH is about pH 6.0.

To maintain effective pH control, the aPC solution may contain apharmaceutically acceptable buffer. Accordingly, upon freeze-drying, theformulation optionally and preferably comprises a pharmaceuticallyacceptable buffer. Representative buffer systems include Tris-acetate,sodium citrate, and sodium phosphate. More preferred buffer systemsinclude sodium citrate and sodium phosphate. The most preferred bufferis sodium citrate. The preferred molarity of the buffer system is 10 mMto 50 mM. A more preferred molarity of the buffer system is 10 mM to 20mM. The most preferred molarity is 40 mM. The skilled artisan willrecognize that many other buffer systems are available for use in theformulations of the invention.

Similarly, during freeze drying and upon reconstitution, the ionicstrength is controlled to ensure solution state stability. The ionicstrength is generally determined by the salt concentration of thesolution. Pharmaceutically acceptable salts typically used to generateionic strength include but are not limited to potassium chloride (KCl)and sodium chloride (NaCl). The preferred salt in the invention issodium chloride. Preferably, the sodium chloride concentration isgreater than 150 mM. More preferably, the sodium chloride concentrationin the freezing solution is between 150 mM to 1000 mM. For a formulationcontaining 2.5 mg/mL aPC, the more preferable sodium chlorideconcentration in the freezing solution is between 150 mM to 650 mM. Evenmore preferably the sodium chloride concentration in the freezingsolution is between 250 mM to 450 mM. Still even more preferably thesodium chloride concentration in the freezing solution is between 300 mMto 400 mM. The most preferable sodium chloride concentration in thefreezing solution is 325 mM for a formulation containing 2.5 mg/mL aPC.

Similarly, for a formulation containing 5.0 mg/mL aPC, the morepreferable sodium chloride concentration in the freezing solution isbetween 150 mM to 1000 mM. Even more preferably the sodium chlorideconcentration in the freezing solution is between 250 mM to 750 mM.Still even more preferably the sodium chloride concentration in thefreezing solution is between 400 mM to 700 mM. The most preferablesodium chloride concentration in the freezing solution is 650 mM for aformulation containing 5.0 mg/mL aPC.

The ratio of aPC:salt:bulking agent (w:w:w) is an important factor in aformulation suitable for the freeze drying process. The ratio variesdepending on the concentration of aPC, salt selection and concentrationand bulking agent selection and concentration. Particularly, a weightratio of one part aPC to between about 7 to 8 parts salt to betweenabout 5 to 7 parts bulking agent is preferred. More preferred is aweight ratio of one part aPC to between about 7.5 to about 8 parts saltto between about 5.5 to about 6.5 parts bulking agent. Most preferred isa ratio of about 1 part aPC to about 7.6 parts salt to about 6 partsbulking agent.

The preferred salt is sodium chloride at a concentration of 325 mM (fora formulation containing 2.5 mg/mL aPC) and 650 mM (for a formulationcontaining 5.0 mg/mL aPC) and at a ratio of about 1.3:1 with sucrose(w:w). This concentration is high enough to cause the salt tocrystallize during the freezing process, most likely resulting in anamorphous mixture of aPC, sucrose, and citrate that can be lyophilized.Thus, the ionic strength of NaCl at the preferred concentrations of 325mM and 650 mM convey stability to the formulation during thefreeze-dying process.

The invention also provides a process for preparing a stable lyophilizedformulation which comprises lyophilizing a solution comprising aPC and achelating agent, preferably about 2.5 mg/mL aPC, about 15 mg/mL sucrose,about 19 mg/mL NaCl, about 500 μM EDTA Disodium, and a sodium citratebuffer having a pH greater than 5.5 but less than 6.5. Furthermore, theinvention provides a process for preparing a stable lyophilizedformulation which comprises lyophilizing a solution comprising about 5mg/mL aPC, about 30 mg/mL sucrose, about 38 mg/mL NaCl about 1 mM EDTADisodium, and a citrate buffer having a pH greater than 5.5 but lessthan 6.5. Such a lyophilization is conducted by routine methods in theart.

Additionally, the invention provides a method of treating disease statesinvolving intravascular coagulation comprising administration of theformulation.

The aPC is preferably administered parenterally to ensure its deliveryinto the bloodstream in an effective form by injecting the appropriatedose as continuous infusion for about one to about ninety-six hours. Theamount of aPC administered is from about 0.01 mg/kg/hr to about 0.05mg/kg/hr. Alternatively, the aPC will be administered by injecting aportion of the appropriate dose per hour as a bolus injection over atime from about 5 minutes to about 30 minutes, followed by continuousinfusion of the appropriate dose for about twenty-three hours to about47 hours, resulting in the appropriate dose administered over 24 hoursto 48 hours.

The following examples will help describe how the invention is practicedand will illustrate the invention. The scope of the invention is not tobe construed as merely consisting of the following examples.

Preparation 1 Preparation of Human Protein C

Recombinant human protein C (r-hPC) was produced in Human Kidney 293cells by techniques well known to the skilled artisan such as those setforth in Yan, U.S. Pat. No. 4,981,952, the entire teaching of which isherein incorporated by reference. The gene encoding human protein C isdisclosed and claimed in Bang, et al., U.S. Pat. No. 4,775,624, theentire teaching of which is incorporated herein by reference. Theplasmid used to express human protein C in 293 cells was plasmid pLPCwhich is disclosed in Bang, et al., U.S. Pat. No. 4,992,373, the entireteaching of which is incorporated herein by reference. The constructionof plasmid pLPC is also described in European Patent Publication No. 0445 939, and in Grinnell, et al., 1987, Bio/Technology 5:1189-1192, theteachings of which are also incorporated herein by reference. Briefly,the plasmid was transfected into 293 cells, then stable transformantswere identified, subcultured and grown in serum-free media. Afterfermentation, cell-free medium was obtained by microfiltration.

The human protein C was separated from the culture fluid by anadaptation of the techniques of Yan, U.S. Pat. No. 4,981,952. Theclarified medium was made 4 mM in EDTA Disodium before it was absorbedto an anion exchange resin (Fast-Flow Q, Pharmacia). After washing with4 column volumes of 20 mM Tris, 200 mM NaCl, pH 7.4 and 2 column volumesof 20 mM Tris, 150 mM NaCl, pH 7.4, the bound recombinant human proteinC zymogen was eluted with 20 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4.The eluted protein was greater than 95% pure after elution as judged bySDS-polyacrylamide gel electrophoresis.

Further purification of the protein was accomplished by making theprotein 3 M in NaCl followed by adsorption to a hydrophobic interactionresin (Toyopearl Phenyl 650 M, TosoHaas) equilibrated in 20 mM Tris, 3 MNaCl, 10 mM CaCl2, pH 7.4. After washing with 2 column volumes ofequilibration buffer without CaCl2, the recombinant human protein C waseluted with 20 mM Tris, pH 7.4.

The eluted protein was prepared for activation by removal of residualcalcium. The recombinant human protein C was passed over a metalaffinity column (Chelex-100, Bio-Rad) to remove calcium and again boundto an anion exchanger (Fast Flow Q, Pharmacia). Both of these columnswere arranged in series and equilibrated in 20 mM Tris, 150 mM NaCl, SmM EDTA Disodium, pH 7.4. Following loading of the protein, theChelex-100 column was washed with one column volume of the same bufferbefore disconnecting it from the series. The anion exchange column waswashed with 3 column volumes of equilibration buffer before eluting theprotein with 0.4 M NaCl, 20 mM Tris-acetate, pH 6.5. Proteinconcentrations of recombinant human protein C and recombinant aPCsolutions were measured by UV 280 nm extinction E0.1%=1.81 or 1.85,respectively.

Preparation 2 Activation of Recombinant Human Protein C

Bovine thrombin was coupled to Activated CH-Sepharose 4B (Pharmacia) inthe presence of 50 mM HEPES, pH 7.5 at 4° C. The coupling reaction wasdone on resin already packed into a column using approximately 5000units thrombin/mL resin. The thrombin solution was circulated throughthe column for approximately 3 hours before adding 2-amino-ethanol (MEA)to a concentration of 0.6 mL/L of circulating solution. TheMEA-containing solution was circulated for an additional 10-12 hours toassure complete blockage of the unreacted amines on the resin. Followingblocking, the thrombin-coupled resin was washed with 10 column volumesof 1 M NaCl, 20 mM Tris, pH 6.5 to remove all non-specifically boundprotein, and was used in activation reactions after equilibrating inactivation buffer.

Purified r-hPC was made 5 mM in EDTA Disodium (to chelate any residualcalcium) and diluted to a concentration of 2 mg/mL with 20 mM Tris, pH7.4 or 20 mM Tris-acetate, pH 6.5. This material was passed through athrombin column equilibrated at 37° C. with 50 mM NaCl and either 20 mMTris pH 7.4 or 20 mM Tris-acetate pH 6.5. The flow rate was adjusted toallow for approximately 20 min. of contact time between the r-hPC andthrombin resin. The effluent was collected and immediately assayed foramidolytic activity. If the material did not have a specific activity(amidolytic) comparable to an established standard of aPC, it wasrecycled over the thrombin column to activate the r-hPC to completion.This was followed by 1:1 dilution of the material with 20 mM buffer asabove, with a pH of either 7.4 or 6.5 to keep the aPC at lowerconcentrations while it awaited the next processing step.

Removal of leached thrombin from the aPC material was accomplished bybinding the aPC to an anion exchange resin (Fast Flow Q, Pharmacia)equilibrated in activation buffer (either 20 mM Tris, pH 7.4 or 20 mMTris-acetate, pH 6.5) with 150 mM NaCl. Thrombin does not interact withthe anion exchange resin under these conditions, but passes through thecolumn into the sample application effluent. Once the aPC is loaded ontothe column, a 2-6 column volume wash with 20 mM equilibration buffer isdone before eluting the bound aPC with a step elution using 0.4 M NaClin either 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4. Higher volumewashes of the column facilitated more complete removal of thedodecapeptide. The material eluted from this column was stored either ina frozen solution (−20° C.) or as a lyophilized powder.

The anticoagulant activity of aPC was determined by measuring theprolongation of the clotting time in the activated partialthromboplastin time (APTT) clotting assay. A standard curve was preparedin dilution buffer (1 mg/mL radioimmunoassay grade bovine serum albumin[BSA], 20 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN3) ranging in protein Cconcentration from 125-1000 ng/mL, while samples were prepared atseveral dilutions in this concentration range. To each sample cuvette,50 μL of cold horse plasma and 50 μL of reconstituted activated partialthromboplastin time reagent (APTT Reagent, Sigma) were added andincubated at 37° C. for 5 min. After incubation, 50 μL of theappropriate samples or standards were added to each cuvette. Dilutionbuffer was used in place of sample or standard to determine basalclotting time. The timer of the fibrometer (CoA Screener HemostasisAnalyzer, American Labor) was started immediately after the addition of50 μL 37° C. 30 mM CaCl2 to each sample or standard. Activated protein Cconcentration in samples is calculated from the linear regressionequation of the standard curve. Clotting times reported here are theaverage of a minimum of three replicates, including standard curvesamples.

EXAMPLE 1

Intravenous infusion solution stability studies in the vial (˜1-mg/mL)with freshly prepared and filtered 0.9% sodium chloride solution (madeat Eli Lilly and Company) containing 5mM, 10 mM, and 20 mM EDTA Disodiumwere conducted using a rhAPC formulation (made at Eli Lilly and Company;2 mg/mL aPC, 15.2 mg/mL sucrose, 12 mg/mL NaCl, and a citrate bufferhaving a pH greater than 5.5 but less than 6.5). The 1-mg/mL I.V.Solutions of rhAPC Formulation prepared in 0.9% sodium chloride solution(made at Eli Lilly and Company), sterile Water for Injection, USP, andin a 150-mL Abbott PVC I.V. bag of 0.9% Sodium Chloride Injection, USPserved as controls. The C-Terminal Light Chain Variant (by LC/MS)results from these studies are presented in Tables 1A and 1B. TABLE 1AC-Terminal Light Chain Variant Results for ˜1-mg/mL I.V. Solution ofrhAPC Formulation in the Vial with Freshly Prepared 0.9% Sodium ChlorideSolution and sterile Water for Injection, USP (Control), and in 0.9%Sodium Chloride Solution Containing 5 mM, 10 mM, and 20 mM EDTA DisodiumC-Terminal Light Chain Sample Description Variant (%) Initial 1-mg/mLI.V. Solution with 1-149: ND Freshly Prepared 0.9% Sodium ChlorideSolution (Control) 24-Hour 1-mg/mL I.V. Solution with 1-149: 59% FreshlyPrepared 0.9% Sodium Chloride Solution (Control) 24-Hour 1-mg/mL I.V.Solution with 1-149: 19% Sterile Water for Injection (Control) Initial1-mg/mL I.V. Solution with 1-149: ND Freshly Prepared 0.9% SodiumChloride Solution Containing 5 mM EDTA Disodium 24-Hour 1-mg/mL I.V.Solution with 1-149: ND Freshly Prepared 0.9% Sodium Chloride SolutionContaining 5 mM EDTA Disodium Initial 1-mg/mL I.V. Solution with 1-149:ND Freshly Prepared 0.9% Sodium Chloride Solution Containing 10 mM EDTADisodium 24-Hour 1-mg/mL I.V. Solution with 1-149: ND Freshly Prepared0.9% Sodium Chloride Solution Containing 10 mM EDTA Disodium Initial1-mg/mL I.V. Solution with 1-149: ND Freshly Prepared 0.9% SodiumChloride Solution Containing 20 mM EDTA Disodium 24-Hour 1-mg/mL I.V.Solution with 1-149: ND Freshly Prepared 0.9% Sodium Chloride SolutionContaining 20 mM EDTA DisodiumN/A = Not Applicable;ND = Detection Limit is <2%

TABLE 1B C-Terminal Light Chain Variant Results for ˜1-mg/mL I.V.Solution of rhAPC Formulation in Sterile Polypropylene Tubes and in a150-mL Abbott PVC I.V. Bag of 0.9% Sodium Chloride Injection, USP(Control), and in 0.9% Sodium Chloride Solution Containing 5 mM, 1 mM,0.2 mM, 0.04 mM EDTA Disodium C-Terminal Light Chain Sample DescriptionVariant (%) Initial 1-mg/mL I.V. Solution in a 150-mL 1-149: 6% PVC I.V.Bag of 0.9% Sodium Chloride Solution (Control) 24-Hour 1-mg/mL I.V.Solution in a 150-mL 1-149: 78% PVC I.V. Bag of 0.9% Sodium ChlorideSolution (Control) 24-Hour 1-mg/mL I.V. Solution in a 150-mL 1-149: NDPVC I.V. Bag of 0.9% Sodium Chloride Solution Containing 5 mM EDTADisodium 24-Hour 1-mg/mL I.V. Solution in a 150-mL 1-149: ND PVC I.V.Bag of 0.9% Sodium Chloride Solution Containing 1 mM EDTA Disodium24-Hour 1-mg/mL I.V. Solution in a 150-mL 1-149: ND PVC I.V. Bag of 0.9%Sodium Chloride Solution Containing 0.2 mM EDTA Disodium 24-Hour 1-mg/mLI.V. Solution in a 150-mL 1-149: ND PVC I.V. Bag of 0.9% Sodium ChlorideSolution Containing 0.04 mM EDTA DisodiumN/A = Not Applicable;ND = Non-detected.Detection Limit is <2%

EXAMPLE 2

Intravenous infusion solution stability studies of a 200-μg/mL rhAPCI.V. solutions in a 150-mL B. Braun/McGaw PAB® I.V. Bag of 0.9% SodiumChloride Injection, USP (Control) and in 150-mL B. Braun/McGaw PAB® I.V.Bags of 0.9% Sodium Chloride Injection, USP containing 20 μM, 50 μM, and100 μM, respectively, of EDTA Disodium were conducted using a rhAPCformulation (made at Eli Lilly and Company; 2 mg/mL aPC, 15.2 mg/mLsucrose, 12 mg/mL NaCl, and a citrate buffer having a pH greater than5.5 but less than 6.5). The rhAPC concentration, potency, and pH resultsfrom these studies are presented in Table 2A. The LC/MS results fromthese studies are presented in Table 2B. TABLE 2A rhAPC Concentration,Potency and pH Results for ˜200-μg/mL I.V. Solutions of rhAPCFormulation in a 150-mL PAB ® I.V. Bag of 0.9% Sodium ChlorideInjection, USP (Control), and in 150-mL PAB ® I.V. Bags of 0.9% SodiumChloride Injection, USP Containing 20 μM, 50 μM, and 100 μM EDTADisodium rhAPC Sample Description Conc. Potency pH Initial 200-μg/mLI.V. Solution of rhAPC 210 μg/mL 456 U/mg 5.98 Formulation in a 150-mLPAB ® I.V. Bag of 0.9% NaCl Inj. (Control)  8-Hour 200-μg/mL I.V.Solution of rhAPC 210 μg/mL 338 U/mg ND Formulation in a 150-mL PAB ®I.V. Bag of 0.9% NaCl Inj. (Control) 12-Hour 200-μg/mL I.V. Solution ofrhAPC 210 μg/mL 315 U/mg ND Formulation in a 150-mL PAB ® I.V. Bag of0.9% NaCl Inj. (Control) 24-Hour 200-μg/mL I.V. Solution of rhAPC 210μg/mL 249 U/mg 6.03 Formulation in a 150-mL PAB ® I.V. Bag of 0.9% NaClInj. (Control) Initial 200-μg/mL I.V. Solution of rhAPC 190 μg/mL 464U/mg* 6.00 Formulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj.Containing 20 μM EDTA Disodium  8-Hour 200-μg/mL I.V. Solution of rhAPC190 μg/mL 450 U/mg* ND Formulation in a 150-mL PAB ® I.V. Bag of 0.9%NaCl Inj. Containing 20 μM EDTA Disodium 12-Hour 200-μg/mL I.V. Solutionof rhAPC 190 μg/mL 434 U/mg* ND Formulation in a 150-mL PAB ® I.V. Bagof 0.9% NaCl Inj. Containing 20 μM EDTA Disodium 24-Hour 200-μg/mL I.V.Solution of rhAPC 190 μg/mL 422 U/mg* 6.01 Formulation t in a 150-mLPAB ® I.V. Bag of 0.9% NaCl Inj. Containing 20 μM EDTA Disodium Initial200-μg/mL I.V. Solution of rhAPC 210 μg/mL 479 U/mg 5.99 Formulation ina 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj. Containing 50 μM EDTA Disodium 8-Hour 200-μg/mL I.V. Solution of rhAPC N/A 461 U/mg ND Formulation ina 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj. Containing 50 μM EDTA Disodium12-Hour 200-μg/mL I.V. Solution of rhAPC N/A 465 U/mg ND Formulation ina 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj. Containing 50 μM EDTA Disodium24-Hour 200-μg/mL I.V. Solution of rhAPC 220 μg/mL 428 U/mg 6.00Formulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj. Containing 50μM EDTA Disodium Initial 200-μg/mL I.V. Solution of rhAPC 210 μg/mL 498U/mg 5.98 Formulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj.Containing 100 μM EDTA Disodium  8-Hour 200-μg/mL I.V. Solution of rhAPCN/A 475 U/mg ND Formulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj.Containing 100 μM EDTA Disodium 12-Hour 200-μg/mL I.V. Solution of rhAPCN/A 470 U/mg ND Formulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj.Containing 100 μM EDTA Disodium 24-Hour 200-μg/mL I.V. Solution of rhAPC220 μg/mL 451 U/mg 6.00 Formulation in a 150-mL PAB ® I.V. Bag of 0.9%NaCl Inj. Containing 100 μM EDTA DisodiumN/A Not ApplicableND = Not Determined*The average of three analyses is reported

TABLE 2B C-Terminal Light Chain Variant Results for ˜200-μg/mL I.V.Solutions of rhAPC Formulation in a 150-mL PAB ® I.V. Bag of 0.9% SodiumChloride Injection, USP (Control), and in 150-mL PAB ® I.V. Bags of 0.9%Sodium Chloride Injection, USP Containing 20 μM, 50 μM, and 100 μM EDTADisodium C-Terminal Light Chain Sample Description Variant (%) Initial200-μg/mL I.V. Solution of 1-149: ND rhAPC Formulation in a 150-mL PAB ®I.V. Bag of 0.9% NaCl Inj. (Control) 24-Hour 200-μg/mL I.V. Solution of1-149: 79% rhAPC Formulation in a 150-mL PAB ® I.V. Bag of 0.9% NaClInj. (Control) Initial 200-μg/mL I.V. Solution of 1-149: ND rhAPCFormulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj. Containing 20μM EDTA Disodium 24-Hour 200-μg/mL I.V. Solution of 1-149: ND rhAPCFormulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj. Containing 20μM EDTA Disodium Initial 200-μg/mL I.V. Solution of 1-149: ND rhAPCFormulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj. Containing 50μM EDTA Disodium 24-Hour 200-μg/mL I.V. Solution of 1-149: ND rhAPCFormulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj. Containing 50μM EDTA Disodium Initial 200-μg/mL I.V. Solution of 1-149: ND rhAPCFormulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj. Containing 100μM EDTA Disodium 24-Hour 200-μg/mL I.V. Solution of 1-149: ND rhAPCFormulation in a 150-mL PAB ® I.V. Bag of 0.9% NaCl Inj. Containing 100μM EDTA DisodiumN/A = Not Applicable;ND = Non-detected.Detection Limit is <2%

EXAMPLE 3

A stable lyophilized formulation is made by lyophilizing a solutioncomprising 2.5 mg/mL aPC, 15 mg/mL sucrose, 19 mg/mL NaCl, 500 μM EDTADisodium, and a sodium citrate buffer having a pH greater than 5.5 butless than 6.5. Also, a stable lyophilized formulation is made bylyophilizing a solution comprising 5 mg/mL aPC, 30 mg/mL sucrose, 38mg/mL NaCl, 1 mM EDTA Disodium, and a citrate buffer having a pH greaterthan 5.5 but less than 6.5.

EXAMPLE 4

A stable lyophilized formulation is made by lyophilizing a solutioncomprising 2.5 mg/mL aPC, 15 mg/mL sucrose, 19 mg/mL NaCl, and a sodiumcitrate buffer having a pH greater than 5.5 but less than 6.5. Also, astable lyophilized formulation is made by lyophilizing a solutioncomprising 5 mg/mL aPC, 30 mg/mL sucrose, 38 mg/mL NaCl, and a citratebuffer having a pH greater than 5.5 but less than 6.5. Prior to freezedrying and upon reconstitution, the pH is maintained in the range of 5.5to 6.5. When the lyophilized formulation is reconstituted, thereconstitution diluent contains a sufficient amount of EDTA Disodium toprovide 100 μM in the reconstituted solution. Alternatively, thereconstituted formulation is added to a diluent suitable foradministration to a patient, such as an intravenous infusion solution,containing a sufficient amount of EDTA Disodium to provide 100 μM in theinfusion solution.

1. A pharmaceutical composition comprising activated protein c and achelating agent.
 2. The composition of claim 1 wherein thepharmaceutical composition is a lyophilized formulation
 3. Thecomposition of claim 2 further comprising a bulking agent.
 4. Thecomposition of claim 3 wherein the bulking agent is selected from thegroup consisting of mannitol, trehalose, raffinose, and sucrose, andmixtures thereof.
 5. The composition of claim 4 further comprising abuffer selected from the group consisting of Tris-acetate, sodiumcitrate, sodium phosphate, and combinations thereof.
 6. The compositionof claim 5 further comprising a buffer such that upon reconstitution theformulation has a pH of about 5.5 to about 6.5.
 7. The composition ofclaim 6 further comprising a salt.
 8. The composition of claim 7 whereinthe salt is selected from the group consisting of potassium chloride andsodium chloride.
 9. A pharmaceutical composition comprising activatedprotein C, a diluent, and a chelating agent.
 10. The composition ofclaim 9 wherein the pharmaceutical composition is a lyophilizedformulation.
 11. The composition of claim 9 wherein the diluent is areconstitution diluent.
 12. The composition of claim 9 wherein thediluent is an intravenous infusion solution.
 13. The composition ofclaim 9 wherein the chelating agent is present in the diluent.
 14. Thecomposition of claim 10 further comprising a bulking agent.
 15. Thecomposition of claim 14 wherein the bulking agent is selected from thegroup, consisting of mannitol, trehalose, raffinose, sucrose, andmixtures thereof.
 16. The composition of claim 15 further comprising abuffer selected from the group consisting of Tris-acetate, sodiumcitrate, sodium phosphate, and combinations thereof.
 17. The compositionof claim 16 further comprising a buffer such that upon reconstitutionthe formulation has a pH of about 5.5 to about 6.5.
 18. The compositionof claim 17 further comprising a salt.
 19. The composition of claim 18wherein the salt is selected from the group consisting of potassiumchloride and sodium chloride.
 20. A process for preparing a lyophilizedformulation of aPC, which comprises freeze drying a pharmaceuticalformulation containing activated protein C and a chelating agent.
 21. Aprocess for preparing a lyophilized formulation of aPC, which comprisesfreeze drying a pharmaceutical formulation containing activated proteinC, a bulking agent, and a chelating agent.
 22. A process of preparing apharmaceutical solution of aPC, which comprises reconstituting alyophilized formulation containing activated protein C with a diluentcontaining a chelating agent.
 23. A process of preparing apharmaceutical solution of aPC, which comprises reconstituting alyophilized formulation containing activated protein C and a bulkingagent with a diluent containing a chelating agent.
 24. A method oftreating a patient in need thereof which comprises administering to thepatient the pharmaceutical composition of any one of claims 1 through19.
 25. (canceled)